The Mir17∼92 Cluster Is an Immunomodulator in CLL Regulating Distinct Functional Responses to Toll-Like Receptors in Subsets with Stereotyped Antigen Receptors

Abstract 3862

Subgroups of CLL cases defined on the basis of the immunogenetic features of their B cell receptors (BcRs), especially those cases belonging to subsets with stereotyped BcRs, exhibit differential responses to immune stimulation through either the BcR or the Toll-Like Receptors (TLRs). This indicates distinct, subset-biased modalities of BcR collaboration with specific TLRs that may extend to the control of cell proliferation or apoptosis, B cell anergy and/or TLR tolerance. In both normal and CLL B cells, the final signaling outcome can be controlled by microRNAs (miRNAs) targeting critical signaling molecules downstream of the BcR and/or the TLRs. With this in mind, here we investigated the impact of immune signaling on miRNA expression and function. We used two paradigmatic stereotyped subsets with distinct molecular and clinicobiological figures: (1) subset #1: unmutated (U) IGHV1/5/7-IGKV1(D)-39 IgM BcRs, aggressive disease, the largest U-CLL subset; and, (2) subset #4: mutated (M) IGHV4–34/IGKV2–30 IgG BcRs, indolent disease, the largest M-CLL subset. We have recently shown that these two subsets show differential functional outcomes after TLR stimulation with subset #4 selectively responsive to TLR1/2 but not to TLR7 stimulation, thus contrasting subset #1 which shows the opposite profile. Based on the above, we cultured negatively selected CD19⁺ B cells from subset #1 and subset #4 cases under the following conditions: (1) non stimulated; (2) stimulated through the BcR with anti-IgM and anti-IgG, respectively; (3) stimulated through TLR7 (Imiquimod) for subset #1, or through TLR1/2 (Pam3CSK4) for subset #4; and, (4) BcR/TLR co-stimulated. Using RQ-PCR Arrays, we measured the expression levels of a series of 33 miRNAs relevant for CLL biology with potential targets in BCR and/or TLR signaling pathways. In subset #1, none of the TLR ligands affected miRNA expression. On the contrary, TLR1/2 stimulation in subset #4 significantly (p<0.05) augmented the expression of 13 miRNAs, including all members of the miR17∼92 cluster and miR-15a. BcR stimulation had no effect, while BcR/TLR1/2 co-stimulation affected the expression of only 3 miRNAs, all members of the miR17∼92 cluster. Bioinformatics analysis revealed MAP3K1, MAP2K3, MAPK8, CASP8 and IKBKB as potential targets of the differentially expressed miRNAs in subset #4 cases after TLR1/2 and/or BCR/TLR co-stimulation. The mRNA expression levels of the targets were measured using RQ-PCR and showed significant (p<8b0.05) down-regulation. Western blotting showed down-regulation of the NF-κB inhibitor IKBKB (miR-15a target), the MAP3K1 and MAPK8 kinases and the CASP8 caspase (all miR17∼92 targets). We have also previously shown that both subset #1 and subset #4 are responsive to TLR9 stimulation with CpG, yet in a heterogeneous fashion. Given that, we next evaluated how stimulation with the same ligand can affect miRNA expression in these two paradigmatic stereotyped subsets. This treatment had almost no effect in miRNA expression in subset #4, while in subset #1 it significantly (p<0.05) augmentend the expression of 17 miRNAs, with miR17∼92 cluster members showing the highest up-regulation. In conclusion, we demonstrate selective action of the miR17∼92 cluster in the regulation of immune responses in CLL subsets with distinct molecular features of the clonotypic BcRs. These results raise the possibility that miR17∼92 is an immunomodulator in CLL, which is in line with recent findings that the miR17∼92 cluster regulates the response of U-CLL to TLR9 triggering. The observed effects on the MAPK and NF-κB pathways have important implications for CLL biology and, perhaps, treatment, particularly in light of the clinical efficacy of novel immune signaling inhibitors.